Biomaterials are materials which are designed to interact with and be incorporated by living tissue. Some examples of biomaterials are dental and orthopaedic implants, surgical sutures and bone cement.
Biomaterials are classified according to their behaviour in vivo. Bioinert materials, such as aluminium oxide, stainless steel, zirconia and poly(ether ether ketone) (PEEK), are compatible with the human body and will not be rejected, but do not promote the integration between the material and the living tissue. Resorbable materials are designed to degrade in vivo and be replaced by living tissue. Resorbable materials include inorganic substances such as calcium sulphate and tricalcium phosphate, but also biodegradable organic polymers. Bioactive materials are said to initiate a biological response which leads to a chemical and biological bond between the material and the living tissue. Hydroxyapatite and so called bioglass are some examples of bioactive materials (1.).
Hydroxyapatite, HA, with the chemical formula Ca5(PO4)3OH, is a mineral which closely resembles the calcium phosphate mineral found in bone and teeth. In bone tissue the calcium phosphate crystals exist in the form of rod-shaped crystals with a length of 20-40 nm, 2 nm thick and 2-4 nm wide, surrounded by a collagen network (2.). Due to its bioactive properties, synthetic HA is extensively used in biomaterial applications where an excellent compatibility between the biomaterial and bone tissue is desired. Commercially, synthetic HA is used as an ingredient in bone cements and composite implants, in coatings for dental and orthopaedic implants, and as a bone graft material, among others.
For certain biomaterial applications, it is highly desirable to use nanosized HA with a particle size of 1-100 nm. It is generally considered that the bioactivity of HA is improved if the HA crystals are of a similar size and shape as those produced by the human body. The body recognizes the nanosized HA as a part of its own bone tissue and starts to grow new bone around the foreign object. For implants, a coating with nanosized HA will significantly increase the bone cell activity compared to microsized HA (3.). For HA/polymer composites, the bioactivity as well as the strength is greatly improved with nanosized HA (4.).
Syntheses of Nanosized Calcium Phosphates, in Particular HA, in Powder Form.
Synthetic HA is commonly produced by an aqueous precipitation method. This can be done by mixing a soluble calcium salt, such as calcium nitrate, in water together with phosphoric acid, with a Ca/P ratio of 5/3. Crystallization is then induced by raising the pH of the solution, for example with ammonium hydroxide:5Ca(NO3)2+3H3PO4+10NH4OH→Ca5(PO4)3OH+9H2O+10NH4NO3 
The immense number of calcium phosphate nuclei which will form immediately after the addition of ammonium hydroxide are typically a few nanometers across. With time, the system will shift toward fewer and larger particles. There are several reasons for this phenomenon. Since the solubility is higher for smaller crystals, there is a tendency for small crystals to dissolve and to crystallize on bigger ones. Small nuclei can also collide and agglomerate to larger crystals, which also results in an average larger crystal size. The driving force is to decrease the overall free surface energy of the system (5.). Due to these effects, a crystallisation done at ambient temperature with calcium nitrate, phosphoric acid and ammonia usually gives a HA powder with a specific surface area of 5-10 m2/g. With a more careful control of the crystallisation parameters, such as pH, concentration, precursor salts, temperature and aging time, it is possible to achieve HA powder with specific surface areas between 40-60 m2/g. The crystal size can also be decreased further by grinding, whereby the crystals are crushed to even smaller sizes (6.).
With additives present in the crystallising solution it is possible to alter the crystallisation process. Additives can adsorb to the crystal surface and prevent dissolution of small crystals, and also shield the crystals from colliding. These effects can shift the crystal size distribution towards smaller crystals. Additives, such as for example poly (ethylene glycol) (7.) and ethanol (8.) have been shown to affect the crystal growth of HA.
Surfactants can also be used as crystal growth inhibitors, since they adsorb on surfaces and therefore can stabilize small crystals. Using surfactant self-assembly methods, it is possible to obtain high specific surface areas of HA. A microemulsion is one example of a self-assembling surfactant system, consisting of nanometer-sized surfactant spheres with water on the inside and a hydrophobic solvent on the outside. When crystallisation is initiated inside the water domains, the microemulsion droplets act as uniformly sized reaction vessels, which protect the precipitated crystal nuclei from further agglomeration and thus yield crystals in the nanometer range (9.).
Bose et al. describes a method to synthesize hydroxyapatite with micro-emulsions, giving a hydroxyapatite powder with a specific surface area of 130 m2/g (10.).
Depending on the surfactant type, other self-assembly structures may form, such as liquid crystalline phases. A liquid crystalline phase is a self-assembly structure which can be created with high concentration ratios of surfactant/water. Compared to a microemulsion, a liquid crystalline phase is a more rigid structure which more effectively will prevent the aqueous domains from colliding. Using liquid crystalline phases it is possible to synthesize HA crystals with a specific surface area of 150-300 m2/g (11.) Additives, such as stearic acid, in combination with synthesis in liquid crystalline phases has also been examined. If stearic acid is present in the water domains of the liquid crystalline phase, the carboxylate group will adhere to the surface of the HA crystals and thereby prevent the crystals from agglomeration. The result is a powder with HA crystals encapsulated by a layer of stearic acid. Upon heat treatment this produces a HA powder of calcium carbonate encapsulated HA crystals. (12.).
The main disadvantage with the methods based on surfactant self-assembly is the relatively low yield. The weight ratio of surfactant/HA crystals is often 100/1. This makes large scale synthesis of nanocrystalline HA powder a cumbersome process, since extensive filtering equipment is needed to purify the crystals and the surfactants used in the process has to be taken care of. There is thus a demand for methods that makes it possible to efficiently produce nanosized crystals of HA.
Syntheses of Nanosized Calcium Phosphates, in Particular HA, as Coatings.
There exists a number of methods to coat implants with HA. The plasma spray technique for example, uses high temperatures to evaporate a HA layer on the implant. The result is a relatively thick layer, usually 60-80 μm, of calcium phosphate which adheres strongly to the substrate (13.). Due to the high temperatures in the plasma, as high as 30 000° C., the calcium phosphate has a high amorphous content (14.). This technique produces uneven coatings on porous implants, and with such a thick layer of HA it is not possible to retain the original surface structure of the substrate.
With the sol-gel technique it is possible to use wet application methods, such as dip-coating, to apply a thin layer of calcium phosphate on implant surfaces. However, this technique requires high temperatures, usually approximately 800° C., or long exposure times at such elevated temperatures to obtain a high crystallinity of the HA layer and is therefore unsuitable to use on titanium implants. It is possible to obtain a thin layer with a Simulated Body Fluid (SBF) immersion technique, wherein the implant is immersed in a SBF for a prolonged period, during which HA crystallizes on the implant surface (15.) The SBF technique works at room temperature but demands long exposure times (at least 24 hours) for a stable HA layer to precipitate on the surface.
The surfactant mediated technique described in EP1781568 (11.) makes it possible to coat the implants with a very thin (5-10 nm) layer of calcium phosphate. The heat treatment step is performed at 500° C. for 5 minutes. This temperature is needed to ensure the decomposition of remaining surfactants on the substrate.
Nishimura et al. (16.) describe a two-step technique to coat surfaces with HA nanocrystals. The substrate is initially coated with an alkoxide, such as aminopropyl-trimethoxysilane. Subsequently, the substrate is dipped into a solution of nanocrystalline HA particles, which creates a layer of HA particles on the surface. The disadvantage with this method is the two-step procedure wherein the substrate must be treated with an alkoxide prior to the attachment of the HA crystals. There is a demand for a one-step method of coating a solid surface with HA nanoparticles.
Surface-Modified HA Crystals.
As already mentioned, a powder with HA crystals encapsulated by a layer of stearic acid has been produced, which upon heat treatment gave a HA powder of calcium carbonate encapsulated HA crystals. (12.).
Stupp et al (17.) describe a technique to adhere polymerized amino acids such as poly(L-lysine), to calcium phosphate crystals. The poly(amino acid)-calcium phosphate aggregates are then attached to a metal surface which has previously been seeded with a layer of calcium phosphate crystals. The result is a layer, usually in the micrometer range, of calcium phosphate crystals dispersed in a network of poly(amino acid) fibers. The average size of the individual crystals is 100-500 nm.
Gonzalez-McQuire et al. (18.) describe a technique to produce HA with attached amino acids. The crystallisation is done by mixing Ca(NO3)2, ammonium phosphate and amino acid with molar ratios of Ca:P:amino acid of 3:1:6 at pH 9 and at a temperature of 80° C. This method creates agglomerated nanosized surface-modified HA crystals.
However, technical solutions are still sought for the need to find techniques to produce nanosized calcium phosphate particles and crystals that have better feasibility and provide HA particles of a small size and high specific surface area.